Fluorescence fluorimetric detector

Fluorescence detectors, discussed in Chapter 1, are extremely sensitive picogram quantities of sample can sometimes be detected. However, most polymers (with the exception of certain proteins) are not fluorescent and thus these detectors are rarely used in GPC. Proteins, particularly those containing tryptophan, fluoresce intensely and are readily detected. Because both the IR and the fluorimetric detector are selective for certain functional groups, rather than being sensitive to analyte mass, there are many pitfalls in quantitation. These and other detectors have been reviewed.177178... 

One of the major disadvantages of HPLC, despite all of the progress in the past years, is a serious lack of detectors, particularly universal detectors, which can match the sensitivity of GC detectors. The best HPLC detectors currently available are (spectro) photometric and (spectro) fluorimetric detectors. It seems logical, therefore, to solve the immediate detection problems at least partially by UV- and fluorescence-derivatization techniques. Recent activities by several research groups show an increased interest in such an approach. Derivatization techniques are not restricted to these two detection modes. Reaction products which yield good MS signals (a favourable fractionation pattern) are... 

At excitation wavelengths and concentration ranges where the simple absorbance fluorescence is linear with concentration, the fluorimetric detector is susceptible to the usual interferences that hinder fluorescence measurements, mainly background fluorescence and quenching. 

Luminescence affords a very sensitive means of detection in flowing systems such as HPLC, electrophoresis, flow injection, and flow cytometry. HPLC fluorescence detectors are similar in operation to conventional fluorimeters. Most fluorescence detectors use filters for crude monochromation. Filters pass light in a wider band than do monochromators. This favors spectral sensitivity because more light excites the sample and is collected by the detector. Grating monochromators, on the other hand, favor selectivity. The fluorimetric detector is susceptible to the usual interferences that hinder fluorescence measurements, namely, background fluorescence and quenching. 

The intense fluorescence of LSD provides the basis for a very sensitive and selective detection of this compound - 10 pg. The excitation wavelength used is about 325 nm 3 21,28,53 and the emission is measured at 389 nm 3, 400 nm 3 or 420 nm 3 3. Prolonged irradiation - 5 min - of LSD trapped in a scanning fluorimetric detector, results in the conversion into a non-fluorescent lumi-derivative. The disappearance of the fluorescence upon... 

Fluorescence detection has found wide application in the analysis of ergot alkaloids 36,50 ScQtt and Lawrence50 found that for the fluorimetric detector used in their investi-... 

A variety of analytical methods has been used for determining trace concentrations of PAHs in environmental samples (Table 6-2). These include GC with various detectors, HPLC with various detectors, and TLC with fluorimetric detectors. Various detection devices used for GC quantification include FID, MS, Fourier transform infrared spectrometer (FT-IR), laser induced molecular fluorescence detector (LIMF), diode array detector (DAD), and gas phase fluorescence detector (GPFDA). GC/MS and HPLC with UV or spectrofluorimetric detectors are perhaps the most prevalent analytical methods for determining concentrations of PAHs in environmental samples. 

The performance of fluorimetric detectors can also be enhanced using FI techniques. The flow-cells of such detectors used for HPLC may readily be adapted for use in FI systems. The reproducibility of fluorescence measurements are reponedly improved by better control of the reaction conditions in the FI systems, and selectivity may be enhanced by on-line removal of potential quenching interferents using FI separation techniques. 

Reductive amination is probably the method most widely used today for the introduction of fluorescent groups into sugars prior to analysis by chromatographic or electrophoretic methods. The fluorescent derivatives thus produced can also be detected by UV absorption, and this is sometimes preferred where the analytes are present at higher concentrations as, in contrast to fluorimetric detectors, the response of UV detectors remains linear over a wide range. However, if only trace amounts are to be analyzed, fluorimetry is the method of choice, as in most cases the derivatives are detectable at picomolar or even femtomolar levels under these conditions. 

Although tryptamine derivatives of isocyanates are both fluorimetrically and amperometrically active, fluorescence detection is preferred because of the higher stability and selectivity of the fluorimetric detector. All isocyanates except HDI can be detected at concentrations as low as 0.5ngml in 1201 air samples. 

As example, Figure 5.18 shows an SIA system with a fluorimetric detector for the determination of aluminum with sodium 8-hydroxyquinoline sulfate [7]. Another example is an in-syringe dispersive liquid—liquid microextraction system for aluminum determination in seawater exploiting a special designed fluorescence detector and flow cell [8]. The detection cell comprised a glass mbe of 3 mm... 

In some cases, analytes should be transformed (by means of derivatization) to compounds with better analytical features for the analytical technique to be used, for instance, gas chromatography requires that low-volatile analytes be transformed into volatile derivatives. Also, the formation of a complex sometimes enables coloured or fluorescent derivatives to be obtained before determination with spectrophotometric or fluorimetric detectors. Other reactions, like hydrolysis, saponification or redox are seldom applied (Figure 2.2.5). 

The chromatographie system eonsisted of a quaternary pump, an automatic sample injector (Hewlett-Packard 1050 Series, Palo Alto, CA, USA) and a programmable fluorimetric detector (Hewlett-Packard, 1046 Series). The detector was linked to a data system (Hewlett-Paekard, HPLC ChemStation) for data acquisition and calculation. According to the results of preliminary experiments obtained by injecting directly into the analytical column standard solutions of PAHs, the deteetor was programmed to measure the fluorescence intensity at the exeitation/emission wavelengths pairs listed in Table 1. 

The main advantage of fluorescence techniques is their sensitivity and measurements of nanogram (10—9 g) quantities are often possible. The reason for the increased sensitivity of fluorimetry over that of molecular absorption spectrophotometry lies in the fact that fluorescence measurements use a non-fluorescent blank solution, which gives a zero or minimal signal from the detector. Absorbance measurements, on the other hand, demand a blank solution which transmits most of the incident radiation and results in a large response from the detector. The sensitivity of fluorimetric measurements can be increased by using a detector that will accurately measure very small amounts of radiation. 

Carbamates and substituted ureas are a numerous group of pesticides widely used to control weeds, pests, and diseases in fruit trees, vegetables, and cereals. Carbamate residues in foods are commonly extracted with water-miscible solvents and determined by using a liquid chromatograph equipped with a sensitive detector, frequently a UV detector. In addition, to obtain adequate detection selectivity, the postcolumn fluorimetric labeling technique is used for methyl carbamates. Substituted ureas are normally extracted from foods with organic solvents, and they can be determined directly by HPLC-UV or after postcolumn derivatization by fluorescence determination of their derivatives. 

The oxidation detector for the fluorimetric analysis of carbohydrates in effluents from liquid chromatography columns provides a sensitive method of analysis in blood and urine [110]. The principle involves the reduction of cerium(IV) to cerium(III) by oxidizable compounds such as organic acids and many carbohydrates. The fluorescence... 

Mukhtar et al. [229] have described a laser fluorimetric method for the determination of uranium (and thorium) in soils. To determine uranium (and thorium) in soils, fluorescent X-rays were measured by the use of a germanium planar detector and chromometric techniques [227]. No sample preparation was required in this method. 

Likewise, the luminescence properties of many analytes can be altered in the presenoe of surfactant aggregates (4,7.,8.). Consequently, addition of micelle-forming surfactants (present either in the LC mobile phase or added post-column) can improve the sensitivity of fluorimetric LC detectors (49,482). Micellar spray reagents have been utilized to enhance the fluorescence densitometric detection of dansylamino acids or polycyclic aromatic hydrocarbons (483). The effect was observed for TLC performed on cellulose or polyamide stationary phases with the micellar spray reagent being either CTAC, SB-12, or NaC (483). More recently, use of nonionic Triton X-100 has been found to improve the HPLC detection of morphine by fluorescence determination after post-column derivatization (486) as well as improve the N-chlorination procedure for the detection of amines, amides, and related compounds on thin-layer chromatograms (488). 

Quantitative fluorimetric analysis on real samples usually requires a separation of the fluorescent analyte from its matrix because of the wide occurrence of fluorescent and quenching impurities. Occasionally, simple solvent extraction will do the trick. More often, chromatographic separation is necessary, and the fluo-rimeter becomes merely a detector for the chromatographic eluates. Recently, however, the application of immunochemistry to analytical chemistry has permitted in-situ analysis on a scale never before possible. Although the earliest immunochemical analyses utilized radioisotopes, the most popular ones used today employ fluorescent probes or labels. 

Fluorimetry was considered in the 1950s as the natural detector for pharmaceuticals, due to its improved selectivity and sensitivity compared with UV-Vis absorption. Recent FIA applications include the determination of diazepam, nitrazepam, and oxazepam in pharmaceutical formulations using acidic hydrolysis and fluorimetric detection. Oxidation with Ce(IV) and measurement of the fluorescence from the released Ce(III), which can be considered as a classical strategy, is an appropriate technique for mixtures of amoxycillin and clavulanic acid where kinetic data are used in combination with partial least-squares multivariate calibration. 

Estimation of true vitamin E in foods requires quantitative determination of all its components since they vary in their biological potency. This vitamin consists of four tocopherols (a, jS, y, and 6) and four tocotrienols (a, jS, y, and d), but the three major constituents responsible for vitamin E activity are the a-, jS-, and y-tocopherols. While these compounds are fluorescent, their esters must be reduced to free alcohols for total tocopherol assays. Total vitamin E can be directly obtained through fluorimetry, but the determination of individual components is carried out using LC with fluorimetric detection. This procedure has been used to determine the composition of vitamin E in seed oils from maize, olives, soya beans, sesame, safflower, and sunflower by measuring the content of all the four tocopherols plus a-tocotrienol. The simultaneous determination of tocopherols, carotenes, and retinol in cheese has been carried out using LC with two programmable detectors coimected in series, a spectrophotometer and a fluorimeter. Carotenes have been determined photometrically, and fluorimetric measurements have been obtained for tocopherol and retinol. 

For routine HPLC analysis, the detection of flavins is carried out either spectro-photometrically, using variable- or fixed-wavelength HPLC detectors in the ultraviolet (e.g., 254 nm) or visible (e.g., 405 nm) region, or fluorimetrically. For riboflavin, the excitation wavelength for fluorimetric detection is usually 440 to 450 nm, and the emission wavelength 530 nm. The detection limit for fluorescence detectors is >1 pmol (0.38 ng) riboflavin, whereas <30 pmol (11 ng) can be detected spectrophotometrically at 254 nm. Photodiode array detectors are significantly less sensitive than normal HPLC spectrophotometers (38). 

The analytical methods proposed for acesulfame-K, cyciamate, and saccharin determination in foods, drinks, dietary products, and pharmaceuticals can be grouped into methods for the determination of an individual artificial sweetener [21-27] and multianalyte approaches [28-38], sometimes also including other sweeteners and/or other food additives, such as colorants or preservatives [39-43]. High-performance liquid chromatography (HPLC) is the most frequently used technique for the determination of these sweeteners, and this is selected by international standard methods because of its multianalyte capability, compatibility with the physicochemical properties of sweeteners, high sensitivity, and robustness [44-47]. However, cyciamate requires chemical derivatization to make it detectable by the most commonly employed UV-absorption detector due to a lack of a chromophore, by conversion to dichlorohexylamine for UV detection or to a fluorescence derivative for fluorimetric detection. Another alternative for cyciamate detection is the postcolumn ion-pair extraction where the eluted sweetener is mixed with an appropriate dye (methyl violet or crystal violet), being detected by visible absorption. Furthermore, cyciamate can be detected directly by refractive index [4]. For this, few HPLC methods for the concurrent determination of these sweeteners exist and... 

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